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CONSENSUS Statement

EXPERT CONSENSUS DOCUMENT Diagnosis and management of pseudohypoparathyroidism and related disorders: first international Consensus Statement

Giovanna Mantovani1, Murat Bastepe2,47, David Monk3,47, Luisa de Sanctis4,47, Susanne Thiele5,47, Alessia Usardi6,7,47, S. Faisal Ahmed 8, Roberto Bufo9, Timothée Choplin10, Gianpaolo De Filippo11, Guillemette Devernois10, Thomas Eggermann12, Francesca M. Elli 1, Kathleen Freson13, Aurora García Ramirez14, Emily L. Germain-Lee 15,16, Lionel Groussin17,18, Neveen Hamdy19, Patrick Hanna20, Olaf Hiort5, Harald Jüppner2, Peter Kamenický6,21,22, Nina Knight23, Marie-Laure Kottler24,25, Elvire Le Norcy18,26, Beatriz Lecumberri27,28,29, Michael A. Levine30, Outi Mäkitie31, Regina Martin32, Gabriel Ángel Martos- Moreno33,34,35, Masanori Minagawa36, Philip Murray 37, Arrate Pereda38, Robert Pignolo39, Lars Rejnmark 40, Rebecca Rodado14, Anya Rothenbuhler6,7, Vrinda Saraff41, Ashley H. Shoemaker42, Eileen M. Shore43, Caroline Silve44, Serap Turan45, Philip Woods23, M. Carola Zillikens46, Guiomar Perez de Nanclares 38,48* and Agnès Linglart6,7,20,48* Abstract | This Consensus Statement covers recommendations for the diagnosis and management of patients with pseudo hy popar athyroi dism (PHP) and related disorders, which comprise metabolic disorders characterized by physical findings that variably include short bones, short stature, a stocky build, early-onset obesity and ectopic ossifications, as well as endocrine defects that often include resistance to parathyroid hormone (PTH) and TSH. The presentation and severity of PHP and its related disorders vary between affected individuals with considerable clinical and molecular overlap between the different types. A specific diagnosis is often delayed owing to lack of recognition of the syndrome and associated features. The participants in this Consensus Statement agreed that the diagnosis of PHP should be based on major criteria, including resistance to PTH, ectopic ossifications, brachydactyly and early-onset obesity. The clinical and laboratory diagnosis should be confirmed by a molecular genetic analysis. Patients should be screened at diagnosis and during follow-up for specific features, such as PTH resistance, TSH resistance, growth hormone deficiency , hypogonadism, skeletal deformities, oral health, weight gain, glucose intolerance or type 2 diabetes mellitus, and hypertension, as well as subcutaneous and/or deeper ectopic ossifications and neurocognitive impairment. Overall, a coordinated and multidisciplinary approach from infancy through adulthood, including a transition programme, should help us to improve the care of patients affected by these disorders.

*e-mail: gnanclares@ osakidetza.eus; Pseudohypoparathyroidism (PHP) and related dis- molecular defects that impair hormonal signalling via [email protected] orders are associated with a spectrum of abnormal receptors that are coupled, through the α-subunit of the https://doi.org/10.1038/ physical characteristics as well as neurocognitive and stimulatory G protein (Gsα), to activation of adenylyl s41574-018-0042-0 endocrine abnormalities that are caused primarily by cyclase (Fig. 1).

The term PHP (Online Mendelian Inheritance in osteodystrophy (AHO), which includes premature clo- Man (OMIM) #103580 for PHP type 1A (PHP1A), sure of growth plates and short bones, short stature, a #603233 for PHP type 1B (PHP1B) and #612462 for stocky build, ectopic ossifications and other poorly PHP type 1C (PHP1C)) describes disorders that share defined abnormalities. In some patients, the physical common biochemical features of hypoparathyroidism features of AHO might be present in the absence of hor- (that is, hypocalcaemia and hyperphosphataemia) mone resistance. Furthermore, based on the number of that are the result of resistance of target tissues to the AHO features and the extent of ectopic ossifications, biological actions of parathyroid hormone (PTH). In patients might be classified as having pseudopseudohy some cases, resistance to other hormones (such as TSH, poparath yroidism (PPHP; OMIM #612463), progressive gonadotropins, growth hormone-releasing hormone osseous heteroplasia (POH; OMIM #166350) or osteoma (GHRH) and calcitonin) that have receptors coupled cutis. PHP and PPHP were initially described by Fuller (ref.1) (ref.2) via Gsα is observed. Patients with PHP1A and PHP1C Albright and colleagues in 1942 and 1952 , are also characterized by the variable expression of a col- respectively; POH was reported more than five decades lection of physical features, termed Albright hereditary later, in 1994 (ref.3). Other features have been attributed

Author addresses 1Fondazione irCCs Ca’ Granda Ospedale Diabetes, Connecticut Children’s Medical HCFMusP, Faculty of Medicine, university of Maggiore Policlinico, endocrinology unit, Center, Farmington, Ct, usa. sao Paulo, sao Paulo, Brazil. Department of Clinical sciences and 16Department of Pediatrics, university of 33Department of endocrinology, Hospital infantil Community Health, university of Milan, Connecticut school of Medicine, Farmington, universitario Niño Jesús, CiBerobn, isCiii, Milan, italy. Ct, usa. Madrid, spain. 2endocrine unit, Department of Medicine, 17aPHP, Department of endocrinology, Cochin 34Department of Pediatrics, autonomous Massachusetts General Hospital and Hospital (HuPC), Paris, France. university of Madrid (uaM), Madrid, spain. Harvard Medical school, Boston, 18university of Paris Descartes, sorbonne Paris 35endocrine Diseases research Group, Hospital Ma, usa. Cité, Paris, France. La Princesa institute for Health research 3imprinting and Cancer Group, Cancer 19Department of Medicine, Division of (iis La Princesa), Madrid, spain. epigenetic and Biology Program (PeBC), institut endocrinology and Centre for Bone Quality, 36Division of endocrinology, Chiba Children’s d’investigació Biomedica de Bellvitge (iDiBeLL), Leiden university Medical Center, Leiden, Hospital, Chiba, Japan. Barcelona, spain. Netherlands. 37Department of Paediatric endocrinology, 4Pediatric endocrinology unit, Department of 20iNserM u1169, Bicêtre Paris sud, Paris royal Manchester Children’s Hospital, Public Health and Pediatric sciences, university sud – Paris saclay university, Le Kremlin-Bicêtre, Manchester university NHs Foundation trust, of torino, turin, italy. France. Manchester, uK. 5Division of Pediatric endocrinology and 21aPHP, Department of endocrinology 38Molecular (epi)Genetics Laboratory, Bioaraba Diabetes, Department of Pediatrics, university and reproductive Diseases, Bicêtre Paris National Health institute, Hospital universitario of Lübeck, Lübeck, Germany. sud Hospital (HuPs), Le Kremlin-Bicêtre, araba-txagorritxu, vitoria-Gasteiz, alava, spain. 6aPHP, reference Center for rare Disorders of France. 39Department of Medicine, Mayo Clinic, Calcium and Phosphate Metabolism, Platform of 22iNserM u1185, Paris sud – Paris saclay rochester, MN, usa. expertise Paris-sud for rare Diseases and Filière university, Le Kremlin-Bicêtre, France. 40Department of endocrinology and internal OsCar, Bicêtre Paris sud Hospital (HuPs), Le 23uK acrodysostosis patients’ group, Medicine, aarhus university Hospital, aarhus, Kremlin-Bicêtre, France. London, uK. Denmark. 7aPHP, endocrinology and diabetes for children, 24Department of Genetics, reference Centre for 41Department of endocrinology and Bicêtre Paris sud Hospital (HuPs), Le Kremlin- rare Disorders of Calcium and Phosphate Diabetes, Birmingham Children’s Hospital, Bicêtre, France. Metabolism, Caen university Hospital, Caen, Birmingham, uK. 8Developmental endocrinology research Group, France. 42Pediatric endocrinology and Diabetes, school of Medicine, Dentistry and Nursing, 25BiOtarGeN, uNiCaeN, Normandie university, vanderbilt university Medical Center, Nashville, university of Glasgow, Glasgow, uK. Caen, France. tN, usa. 9iPOHa, italian Progressive Osseous 26aPHP, Department of Odontology, Bretonneau 43Departments of Orthopaedic surgery and Heteroplasia association, Cerignola, Hospital (PNvs), Paris, France. Genetics, Center for research in FOP and Foggia, italy. 27Department of endocrinology and Nutrition, related Disorders, Perelman school of Medicine, 10K20, French PHP and related disorders patient La Paz university Hospital, Madrid, spain. university of Pennsylvania, Philadelphia, association, Jouars Pontchartrain, France. 28Department of Medicine, autonomous Pa, usa. 11aPHP, Department of medicine for university of Madrid (uaM), Madrid, spain. 44aPHP, service de Biochimie et Génétique adolescents, Bicêtre Paris sud Hospital (HuPs), 29endocrine Diseases research Group, Hospital Moléculaires, Hôpital Cochin, Paris, France. Le Kremlin-Bicêtre, France. La Paz institute for Health research (idiPaZ), 45Department of Pediatrics, Division of 12institute of Human Genetics, Medical Faculty, Madrid, spain. endocrinology and Diabetes, Marmara rwtH aachen university, aachen, Germany. 30Division of endocrinology and Diabetes and university, istanbul, turkey. 13Department of Cardiovascular sciences, Center for Bone Health, Children’s Hospital of 46Department of internal Medicine, Bone Center Center for Molecular and vascular Biology, Philadelphia and Department of Pediatrics, erasmus MC – university Medical Center Gasthuisberg, university of Leuven, university of Pennsylvania Perelman school of rotterdam, rotterdam, Netherlands. Leuven, Belgium. Medicine, Philadelphia, Pa, usa. 47these co-second authors contributed equally: 14aePHP, spanish PHP and related disorders 31Children’s Hospital, university of Helsinki and Murat Bastepe, David Monk, Luisa de sanctis, patient association, Huércal-Overa, Helsinki university Hospital, Helsinki, Finland. susanne thiele, alessia usardi almería, spain. 32Osteometabolic Disorders unit, Hormone and 48these are co-last authors: Guiomar Perez de 15albright Center & Center for rare Bone Molecular Genetics Laboratory (LiM/42), Nanclares, agnès Linglart; all the other authors Disorders, Division of Pediatric endocrinology & endocrinology Division, Hospital das Clínicas are listed in alphabetical order

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Fig. 1 | Molecular defects in the PTH–PTHrP signalling pathway in PHP pseudo hy pop ar ath yroi dism (PHP) and related disorders are due to and related disorders. Upon ligand binding (parathyroid hormone (PTH) molecular defects within the PTH–PTHrP signalling pathway , with the and parathyroid hormone-related protein (PTHrP) are shown on the figure), exception, perhaps, of ectopic ossification. The diseases caused by the G protein coupled PTH/PTHrP receptor type 1 (PTHR1) activates the alterations in the genes that encode the indicated proteins are shown in heterotrimeric Gs protein. The Gsα subunit triggers the activation of blue boxes. Differential diagnoses are shown in grey boxes. CRE, cAMP adenylyl cyclase, which leads to cAMP synthesis. cAMP then binds to the response element; HDAC4, histone deacetylase 4; G protein, trimer α, β and γ; regulatory 1 A subunits (R) of protein kinase A (PKA), the predominant HTNB, autosomal dominant hypertension and brachydactyly type E effector of cAMP. Upon cAMP binding, the catalytic subunits (C) dissociate syndrome; PHP, pseudohypoparathyroidism; PHP1A , pseudohypoparath from the R subunits and phosphorylate numerous target proteins, including yroidism type 1A ; PHP1B, pseudohypoparathyroidism type 1B; PHP1C, cAMP-responsive binding elements (CREB) and the phosphodiesterases pseudo hy popar ath yroi dism type 1C; POH, progressive osseous (PDEs; such as PDE3A and PDE4D). CREB activates the transcription heteroplasia; PPHP, pseudopseudohypoparathyroidism; PTHLH, of cAMP-responsive genes. Intracellular cAMP is then deactivated parathyroid hormone-like hormone; TF, transcription factor ; TRPS1, by PDEs, including PDE4D and PDE3A. The main clinical features of zinc-finger transcription factor TRPS1.

to these disorders since their identification, such as intra- Acrodysostosis is uncommon; the prevalence of the uterine growth failure, early-onset obesity (that is, devel- disease is unknown, and clinical, biochemical and opment in the first few months of life and full expression radiological features overlap with those of PHP1A before the end of infancy), hypogonadism, growth and PPHP6,11–15. hormone (GH) deficiency and cognitive impairment, A molecular cause can be identified in an estimated including developmental delay and loss of intellectual 80–90% of patients with PHP or related disorders16,17. function4. Acrodysostosis (OMIM #101800) refers to The most common underlying mechanisms are de novo a group of chondrodysplasias that resemble PHP in or autosomal dominantly inherited genetic mutations some patients, owing to the presence of brachydactyly and/or epigenetic, sporadic or genetic-based alterations, and often resistance to PTH and TSH, but differ from within or upstream of GNAS4,18,19, PRKAR1A6, PDE4D5,20 PHP owing to more extensive facial dysmorphism, nasal or PDE3A21 (Fig. 1; Supplementary Table 1). hypoplasia and often developmental delay5,6. In the absence of molecular analysis, the clinical and The exact prevalence of PHP is unknown. Studies biochemical overlap between PHP and related disorders published in 2000 and 2016 estimated the prevalence can lead to challenges in diagnostic classification and to be 0.34 in 100,000 in Japan7 and 1.1 in 100,000 in thus in understanding the natural history of the different Denmark8. A limitation of both studies is that the inves- types. A consensus meeting was organized to develop tigators did not confirm the clinical diagnosis of PHP by recommendations for the diagnosis and management of a molecular analysis for the majority of the patients. The patients with PHP and related disorders. prevalence of POH has never been estimated. However, The European Cooperation in Science and Techno it seems to be extremely rare, as <60 cases have been logy (COST) action BM1208 (European Network for reported worldwide up to December 2016 (refs9,10). The Human Congenital Imprinting Disorders, see Related overlap between PPHP and PHP1A means that determin- links), the European Reference Network on Rare ing the prevalence of PPHP is even more complicated. Endocrine Conditions (ENDO-ERN), the European

Reference Network on Rare Bone Disorders (BOND- A.L., Su.T., S.F.A., G.D.F., L.G., H.J., E.L.N., M.A.L., ERN), the European Calcified Tissue Society (ECTS), the O.M., P.M., L.R., R.R., A.H.S., Se.T., P.W. and M.C.Z.), Asian Pacific Paediatric Endocrine Society (APPES), molecular diagnosis (WG2: G.P.d.N., M.B., D.M., T.E., the European Society of Human Genetics (ESHG), the F.M.E., K.F., P.H., M.-L.K., A.P., E.M.S. and C.S.) and Pediatric Endocrine Society (PES), the European clinical management (WG3: G.M., R.B., T.C., L.d.S., Society of Endocrinology (ESE) and the European Soci G.D., A.G.R., E.L.G.-L., N.H., O.H., P.K., N.K., B.L., ety for Paediatric Endocrinology (ESPE) support this R.M., G.A.M.-M., M.M., R.P., A.R., V.S. and A.U.). Consensus Statement. Preparation for the consensus took >24 months, including two preparatory meetings. A preliminary Methods document summarizing the questions addressed in the Thirty-seven participants from 13 countries were preparatory meetings was prepared by each WG and invited to participate in the development of this shared for review with all the experts before the final Consensus Statement based on their publication record consensus meeting. At the final consensus meeting, and expertise in PHP and related disorders, and seven propositions and recommendations were considered by representatives from patient support groups from four participants and discussed in plenary sessions, enabling countries were also included. Health-care profession- reformulation of the recommendations, if necessary. als included endocrinologists, paediatric endocrinolo- Where published data were unavailable or insufficient, gists, paediatric nephrologists, a neonatologist, dentists, experts’ clinical experiences and opinions were consid- molecular biologists, molecular geneticists and a project ered. Therefore, this Consensus Statement focuses on coordinator/manager. Representatives from patient sup- disorders for which we have sufficient published data port groups (Spanish Association of Pseudohypopara- and/or expertise, including PHP1A, PHP1B, PHP1C, thyroidism (AEPHP), K20, the acrodysostosis group and PPHP, POH and acrodysostosis. All experts voted on the Italian Association of POH (IPOHA)) participated the recommendations proposed by each working group in all the discussions but did not vote for the final rec- using the following system: A. evidence or general ommendations. Experts included representatives nom- agreement allows full agreement with the recommen- inated by the council and clinical practice committees dation; B. evidence or general agreement is in favour of from six international societies, two European reference the recommendation; C. evidence or general agreement networks and a European network on imprinting disor- is weak for the recommendation; D. there is not enough ders (COST BM1208). All participants signed a conflict evidence or general agreement to agree with the recom- of interest declaration, and the consensus was strictly mendation. If there was a majority of D, the recommen- supported by funding from academic or professional dation was not accepted. Depending on the proportion societies only, with no sponsorship from the pharma- of votes received by the option with the most votes, the ceutical industry. A Delphi-like consensus methodology strength of the recommendation was recorded as fol- was adopted22. lows: + (26–49% of the votes), ++ (50–69% of the votes) A comprehensive literature search was conducted and +++ ( ≥70% of the votes). using PubMed including articles published from 1 January 1990 through 18 December 2016. The Clinical diagnosis search terms used were “pseudohypoparathyroidism PHP and related disorders vary considerably in clinical or PHP”, “Albright’s Hereditary Osteodystrophy presentation and disease severity between affected indi- or AHO”, “pseudopseudo hy popar ath yroi dism or viduals, even among patients carrying the same genetic PPHP”, “progressive osseous heteroplasia or POH”, alteration. The clinical symptoms (for instance, ossifi- “acrodysostosis”, “osteoma cutis”, “ectopic ossifica- cations and brachydactyly) and abnormalities that can tions”, “subcutaneous ossifications”, “PTH resistance”, be detected in a laboratory (for instance, hypocalcaemia “brachydactyly”, “Fahr syndrome”, “calcaemia” and and raised levels of PTH) (Table 1) typically worsen dur- “hypocalcaemia”. The search was restricted to patients ing mid and late childhood and are usually unnoticed with a genetic diagnosis, so the following search terms in very young children. A correct diagnosis can thus were also included: “GNAS or GNAS1 or Gs-alpha”, be elusive during infancy and in patients with atypical “PRKAR1A”, “PDE4D”, “ PDE3A”, “ PTH1R or PTHR1”, features23. “HOXD13”, “ HDAC4”, “ TRPS1”, “ PTHLH”, “multi locus hypomethylation or multilocus methylation Clinical definitions defect or multilocus imprinting disturbance or MLID” Albright hereditary osteodystrophy. The term AHO is and “prenatal testing and imprinting”. The year 1990 used to indicate a constellation of physical features orig- was chosen as a cut-off as it was the year in which the inally described by Albright1, including a round face, a first inactivating GNAS mutations associated with stocky habitus with short stature, brachydactyly and PHP were described. Only publications in English ectopic ossification. Short bones are not present at birth were considered. Additional relevant articles on ini- and result from premature closure of the epiphyses, lead- tial clinical descriptions, differential diagnoses and ing to a reduced period of growth. Although all bones tend treatments were also identified by PubMed searches to be short, shortening is most marked acrally (that is, in when supplementary information was necessary. the hands and feet). Subsequently, developmental delay A comprehensive review of >800 articles formed the was added as an additional feature of AHO24,25. Obesity, basis of discussion by three working groups (WGs). particularly early-onset obesity, and macrocephaly relative These groups focused on clinical diagnosis (WG1: to height might be part of AHO26–28.

Table 1 | Main clinical features of PHP and related disorders Feature PHP1A PHP1B PPHP PoH AcrdyS1 AcrdyS2 Growth • Growth velocity • Macrosomia • SGA SGA • SGA • SGA decreasing • Average adult • Growth velocity • Adult short stature • Adult short progressively stature decreasing stature • Adult short stature progressively • Adult short stature Obesity Early onset Early onset Normal weight or Normal weight Present Present lean or lean Brachydactyly 70–80% 15–33% <30% Rare 97% 92% Advanced bone age 70–80% 15–33% Unknown Unknown 100% 100% Ectopic ossification 30–60% 0–40% 18–100% 100% 0% 0% PTH resistance 100% 100% Rare and mild Absent 100% 29% (progressive) TSH resistance 100% 30–100% Rare and mild Absent ~100% 16% Neurological • Neurocognitive Cerebral Unknown Unknown Unknown Neurocognitive symptoms impairment calcifications impairment • Cerebral calcifications Gonads Gonadotropin Normal Normal Unknown Case reports of Unknown resistance anatomical dysfunction Pseudo hypopar athyroi dism (PHP) and related disorders affect many organs unequally. The clinical and biochemical features of the main diseases have been represented with their frequency when known. ACRDYS1, acrodysostosis due to mutation in PRKAR1A; ACRDYS2, acrodysostosis due to mutation in PDE4D; PHP1A , pseudo hypopar athyroi dism type 1A due to maternal loss of function mutation at the GNAS coding sequence; PHP1B, pseudohypoparathyroidism type 1B due to methylation defect at the GNAS coding sequence; POH, progressive osseous heteroplasia (due to paternal loss-of-function mutation at the GNAS coding sequence); PPHP, pseudopseudohypopar athyroi dism (due to paternal loss of function mutation at the GNAS coding sequence); PTH, parathyroid hormone; SGA , small for gestational age.

Pseudohypoparathyroidism. The demonstration that lev- form is considered to be a variant of PHP1A in the text

els of Gsα, the α-subunit of the heterotrimeric G protein and the recommendations unless otherwise specified. that couples heptahelical receptors to activation of adenylyl PHP1B was initially defined as isolated resistance to

cyclase, were reduced in erythrocytes from some patients PTH, absence of AHO and normal levels of Gsα activity. with PHP led to the first subclassification within PHP29. Subsequent analyses have demonstrated that some PHP1A was initially defined as the association of patients with PHP1B display additional features that over- resistance to multiple hormones, including PTH and lap with PHP1A. As in PHP1A, PTH resistance might not 44–50 TSH, features of AHO and decreased Gsα activity using be present at birth and develops only over time . TSH in vitro assays29. Evidence of TSH resistance is often pres- resistance and patterns of excessive intrauterine growth or ent at birth and can lead to the misdiagnosis of congeni- weight gain at birth or during early infancy and childhood tal hypothyroidism24,30, whereas PTH resistance develops have been described44,51,52. Some patients with PHP1B during childhood31–34. Similarly, brachydactyly devel- present with one or several features of AHO, the most ops progressively and usually becomes obvious before frequent being brachydactyly51,53. Subcutaneous ossifica- puberty34,35. Additional features have been described in tions are very uncommon51,53,54. Owing to the overlap of patients with PHP1A. For instance, affected patients are some AHO features, distinguishing between PHP1A and born with a moderately reduced birth length that usually PHP1B can be difficult in some patients; even more so as, 24,30,36–38 does not prompt investigations . Obesity might although rarely investigated, mildly decreased Gsα activity develop in very early life and be recognized before any has also been described in some patients with PHP1B55. endocrine disturbances appear in early childhood30,35,39. PHP type 2 (PHP2) is characterized by an increase in The presence of extensive or progressive ossifications levels of cAMP in response to exogenous PTH infusion that extend deep into connective tissues is unusual but but a deficient phosphaturic response56. The exact molec- does not preclude the diagnosis of PHP1A40,41. Cognitive ular cause of this disease variant is still unknown, but it impairment has been associated with the diagnosis of has been suspected that PHP2 could either be an acquired PHP1A; when present, the severity of impairment is defect secondary to vitamin D deficiency57 or be due to 24,42 highly variable . However, about 30% of patients with defective signalling downstream of Gsα, as in patients PHP1A present with normal cognitive development24. with acrodysostosis due to PRKAR1A mutations6. PHP1C has been defined as the association of all the features of PHP1A but with documentation of normal Pseudopseudo hy popar ath yroi dism. PPHP is defined

Gsα activity in in vitro complementation assays that as AHO, with decreased Gsα activity, in the absence of

do not assess the ability of receptors to activate the Gs PTH resistance. Birthweight and length restriction are heterotrimer43. As a result of the overlap with PHP1A and frequent36. Subcutaneous ossifications are also frequent 58,59 of the lack of assessment of Gsα activity in most reports (osteoma cutis and bony plaques) and highly sugges-

(and thus the lack of distinction between PHP1A and tive of Gsα deficiency. In some cases, mild resistance to PHP1C), we have not specifically addressed PHP1C. This PTH and TSH might occur60.

Progressive osseous heteroplasia. POH is defined by the • Major criterion: short stature by adulthood relative presence of ectopic ossifications that are progressive and to the height of the unaffected parent extend deep into connective tissue. At least one bony • Additional criterion: stocky build plate is present. Patients with POH usually have no • Additional criterion: round face in comparison other features of AHO and have normal responsiveness with siblings and degree of obesity, if present to PTH. The ossifications might lead to severe ankyloses • Additional criterion: ectopic (and often subcutaneous) of affected joints and focal growth retardation. Several ossifications clinical and genetic features can be suggestive of the diag- All features might not be present and might evolve nosis, such as a mutation involving exons 1–13 of the throughout the observation period. Obesity, SGA, paternal GNAS allele, radiographic evidence of a reticular dental manifestations and cognitive impairment are pattern of ossification, histological evidence of exclusive present only in a subgroup of patients; these features intramembraneous ossification or both intramembrane- are not required for the diagnosis of AHO (A++). ous and endochondral ossification, lateralization of the 1.3. Clinical and biochemical major criteria for PHP and ossifications in a dermomyotomal pattern, being born related disorders are as follows (A+++): small for gestational age (SGA), leanness and onset of • PTH resistance ossifications before 1 year of age10. POH-like features • And/or subcutaneous ossifications that can have also been described in several patients in whom the include deeper ossifications GNAS mutations were located on the maternal allele41. • And/or early-onset (before 2 years of age) obesity associated with TSH resistance or with one of the Acrodysostosis. Acrodysostosis is defined as the asso- above ciation of severe brachydactyly, facial dysostosis and • And/or AHO alone nasal hypoplasia. Brachydactyly usually affects all pha- • With or without a family history lanxes, metacarpals and metatarsals except for thumbs 1.4. The following features support the diagnosis of PHP and halluces. On radiographs, epiphyses display a cone and related disorders (A+++): shape, bone age is advanced and abnormalities might • Endocrine: elevated levels of TSH, unexplained be present at birth or soon thereafter. In some cases, the congenital hypothyroidism (mildly elevated lev- chondrodysplasia is indistinct from that of AHO11–13,15,61. els of TSH), hypogonadism, hypercalcitoninaemia Other symptoms might also be present in patients with and/or GH deficiency acrodysostosis, such as cognitive impairment15,62, being • Neurological: cognitive impairment, hearing born SGA and resistance to PTH and/or other hormones impairment, spinal stenosis, Chiari malformation 6,13,15,63,64 that signal through Gsα . type 1, syringomyelia, carpal tunnel syndrome and/or craniosynostosis Effect of correct diagnosis. Confirmation of the diagnosis • Mineralization defects: enamel hypoplasia, delayed is important for patients and for parents of children with tooth eruption or tooth ankylosis, oligodontia or PHP and related disorders, as this guides appropriate hypodontia, advanced skeletal maturation, cataract management and prevents further, sometimes exten- and/or central nervous system (CNS) calcifications sive, investigation of frequent presenting signs, such as • Others: sleep apnoea, ear infection, asthma, growth failure, obesity and/or seizures, to identify the early-onset obesity, SGA and/or cryptorchidism underlying cause. Conversely, exclusion of the diagnosis 1.5. PHP and related disorders are primarily clinical diag- will prompt further investigation and consideration of noses. Identification of the molecular cause should alternative underlying conditions. be performed to confirm the clinical diagnosis and Establishing the correct diagnosis in patients with allow the characterization of the subtype of the PHP, PPHP, POH or acrodysostosis allows an appropri- disease (A++). ate conversation to take place with patients and families, 1.6. Testing for genetic or epigenetic causes should be including anticipatory guidance, and informs decisions based on the clinical characteristics, local access to on biochemical screening and treatment of potential genetic testing and the most likely identified causes endocrine defects. Families can be directed to appropriate of the disease at the time of analysis according to the support groups, and genetic counselling can be offered. algorithm (see Molecular diagnosis section) (A++). Identifying patients with these disorders is also important The experts have highlighted that administration to allow further research into the underlying incidence, of exogenous PTH (modified Ellsworth–Howard natural history and aetiology of the disease phenotypes. test) is not necessary but might be helpful in

research settings. Assessment of Gsα bioactivity is Recommendations usually not required for the clinical diagnosis of 1.1. The diagnosis of PHP and related disorders should PHP and related disorders. be based on clinical and biochemical characteristics, which will vary depending on the age of the patient Main clinical components and, in some cases, on the family history (A+++). In the majority of patients with PHP, the most impor- 1.2. In the context of PHP and related disorders, the tant clinical manifestation is symptoms of hypocalcae- diagnosis of AHO should be based on the presence mia due to PTH resistance (45–80%)65. Periods of rapid of the following clinical features: growth and the associated increased calcium require- • Major criterion: brachydactyly type E (premature ment, or nutritional calcium or vitamin D deficiency, fusion of the epiphyses) might trigger or intensify symptoms34.

Resistance to PTH. In patients with PHP1A, resistance of adipose-derived progenitor mesenchymal stem cells to PTH is usually absent at birth and evolves over life that lead to predominantly web-like intramembranous (from 0.2 years to 22 years)18,31,34, while the clinical ossifications73,74. Unlike fibrodysplasia ossificans progres- manifestations typically occur later. These data sug- siva (FOP), where injury, viral infections and immuniza- gest that PTH resistance begins in early childhood, tions can lead to ossifications, no formal evidence exists and the resultant changes in serum levels of calcium and to suggest that the same events lead to ectopic bone for- phosphorus develop gradually, at some point during mation in GNAS-based conditions. Ectopic ossifications adulthood31,32,66–68. The first biochemical abnormalities are found in 100%, 80–100%, 30–60% and very uncom- to become apparent are elevated serum levels of PTH monly in patients with POH, PPHP or AHO, PHP1A and elevated serum levels of phosphorus, followed by and PHP1B, respectively, and have never been reported hypocalcaemia. When hypocalcaemia is present, urine in patients with acrodysostosis13,61,75–78. levels of calcium are low, whereas calcitriol levels might Osteoma cutis (a form of ectopic ossification) is be either low or normal18. An interval of up to 4.5 years usually present at birth or develops very early in life, usually occurs between the start of the elevation in levels more rarely during childhood and adulthood, as a single of PTH and phosphorus and onset of hypocalcaemia67. large plaque in the skin, as an isolated dermal nodule or Patients with PHP1B might show variable degrees nodules or as multiple papules on the face79. Osteoma of PTH-resistant hypocalcaemia or normocalcaemia, cutis is associated with normal serum levels of calcium despite identical epigenetic changes involving tran- and phosphorus throughout life, which suggests that scription start site (TSS)–differentially methylated the mutation is on the paternal GNAS allele41,58,59,80–82. region (DMR) at exon A/B of GNAS (GNAS A/B:TSS- Ossifications that develop in patients with POH are DMR)47,66,69,70. Long-standing secondary hyperpara progressive and extend into the muscles, tendons and thyroidism with chronic hypocalcaemia and calcitriol ligaments10,41,83. The ossifications in patients with POH deficiency has been associated with tertiary hyperpar- can be limited to or be much more prominent on one athyroidism in these patients71. Chronically elevated side of the body, which suggests that the mechanism of levels of PTH might also lead to bone resorption and the disease encompasses a second mutational mosaic hit 84 demineralization that resemble the bone changes seen or variations in GNAS imprinting or Gsα expression . in rickets in children with irregular and widened metaphyses, Madelung-like deformity or primary hyper- Recommendations parathyroidism, including brown tumours (a bone 1.8. Ectopic ossifications should be considered as a lesion that can occur when osteoclast activity due to specific sign of GNAS mutations (specifically when hyperparathyroidism is excessive)72. observed at birth or in early childhood) (A++). Resistance to PTH is also present in patients with muta- 1.9. The description of the ossifications, their location tions in PRKAR1A5,6,15,20,63,64; however, hypocalcaemia has and their extension in connective tissues should not been documented in these patients yet. Patients with be considered to establish the diagnosis of POH PDE4D mutations usually display normal levels of PTH, versus osteoma cutis, PHP1A, PHP1C, PPHP or except in the context of calcifediol deficiency5,13–15,20,62,63. AHO, where the latter conditions have ectopic bone that remains superficial (B++). Recommendations 1.10. The diagnosis of osteoma cutis, nodules of subcuta- 1.7. The definition of PTH resistance is as follows: neous ossifications or deep heterotopic ossifications • The association of hypocalcaemia, hyperphos- should trigger a clinical and biochemical work-up phataemia and elevated serum levels of PTH in to search for signs of AHO, PTH and TSH resist- the absence of vitamin D deficiency and when ance or FOP, especially if the first digit of both feet is magnesium levels and renal function are normal. abnormal. If the diagnosis is obvious or very likely, a • PTH resistance in the context of PHP and related biopsy sample is not needed and is contraindicated disorders should be suspected when PTH is at, or in case of suspicion of FOP (A+++). above, the upper limit of normal, in the presence of normal calcifediol levels and elevated serum Brachydactyly. In PHP and related disorders, brachy- levels of phosphorus, even in the absence of overt dactyly can be classified as type E, which is defined as hypocalcaemia. variable shortening of the metacarpals with, usually, PTH resistance and consequent changes in serum lev- normal length of phalanges, occasionally accompanied els of calcium, phosphorus and PTH can be variable, by relatively shortened metatarsals. In PHP and related and repeated testing might be required (A+++). disorders, the fifth, fourth and third metacarpal and the first and fourth distal phalanges are the most affected Ectopic ossification. Disorders caused by molecular bones of the hand85; metatarsals are often shortened as alterations of the GNAS gene or locus, such as PHP1A, well. Brachydactyly develops over time and might not be PHP1C, PPHP and POH, can feature ectopic ossification. evident in early life, except in patients with acrodysos- Ectopic ossification is not calcification and is unrelated to tosis6,35,86. The frequency and severity of brachydactyly serum levels of calcium and phosphorus. The ectopic ossi- vary among the different disorders: 70–80% in PHP1A51, 46,51,70,87–93 fications are a manifestation of Gsα deficiency in mesen- 15–33% in PHP1B and all patients with acrodys- chymal stem cells, with de novo formation of extraskeletal ostosis5,6,14,15,20,62–64,94–96 (Table 1). However, brachydactyly is osteoblasts that form islands of ectopic bone in the dermis not specific to PHP and related disorders and can be found and the subcutaneous fat as a result of the differentiation in patients with, for example, tricho–rhino–phalangeal

syndrome, brachydactyly mental retardation syndrome exact prevalence is not known, as the results of objective or Turner syndrome (Table 2). standard tests are rarely reported (40–70% of patients with PHP1A, 0–10% of patients with PPHP or POH, Recommendations rare in patients with PHP1B and of variable prevalence 1.11. When brachydactyly type E is identified, other in patients with acrodysostosis)1,18,23,24,26,42. disorders associated with bone dysplasia should be Elevated levels of calcitonin are found in a large sub- excluded, for example, Turner syndrome, tricho– set of patients with PHP1A and PHP1B, after exclusion rhino–phalangeal syndrome, isolated brachydac- of medullary thyroid carcinoma. Elevated levels of cal- tyly type E and brachydactyly mental retardation citonin might be considered as a marker of hormone syndrome (A+++). resistance in a patient with a PHP or related disorder114,115 1.12. If not present in infancy, brachydactyly type E Resistance to gonadotropins seems to be less severe should be searched for (clinically and radiologi- than resistance to other hormones, such as PTH and cally) from early childhood onwards in all patients TSH; laboratory abnormalities indicating elevated levels with PHP and related disorders (A+++). of luteinizing hormone (LH) or follicle-stimulating hormone (FSH) have been reported by some groups116–121, TSH resistance whereas others could not confirm these findings122–127. Patients with PHP1A frequently (if not always97) present These findings have led to the hypothesis that with raised serum levels of TSH and thyroid hormone patients with PHP1A display only partial resistance to levels that are normal or slightly reduced. Some patients gonadotropins120. present with overt clinical hypothyroidism. Elevated Although nonspecific symptoms such as early-onset levels of TSH due to TSH resistance might be present at obesity, short stature and/or hypothyroidism might be birth and detected on neonatal screening24,30,31,60,67,98–100. present in early infancy or childhood, the diagnosis is In PHP1B, TSH levels are at the high end of normal often delayed or not recognized until early puberty for or mildly elevated in 30–100% of patients47,87,89,92,101–105. patients with PHP1A and acrodysostosis or until ado- TSH resistance is present in patients with acrodysos- lescence or adulthood for patients with PHP1B, unless tosis owing to PRKAR1A mutations but not in those the family history is positive. The presence of ectopic with PDE4D mutations13,15. Thyroid autoantibodies ossifications (which are disease-specific) might trigger are usually absent in patients with PHP1A, PHP1B an earlier diagnosis of POH or osteoma cutis. or acrodysostosis who have elevated levels of TSH. Nevertheless, given the high prevalence of autoimmune Differential diagnoses thyroid disease, the presence of thyroid autoantibodies The differential diagnosis of patients with obesity, does not mean that the patient might not also have TSH early-onset (even congenital) hypothyroidism, short resistance. stature and/or brachydactyly is very broad and includes many endocrine and syndromic diseases (Table 2). Additional frequent clinical features Some features, however, should prompt the clinician Many aspects of PHP and related disorders do not lead to consider diagnoses other than PHP or related dis- to measurable hormonal changes and are not yet rec- orders. These features include additional bone anom- ognized as being part of the spectrum of PHP-related alies, such as syndactylies, exostoses or Madelung disorders (Table 1). deformity128, and/or facial dysmorphism or other SGA has been described in patients with a paternal features suggestive of Turner syndrome or tricho– GNAS mutation (that is, patients with PPHP or POH)106. rhino–phalangeal syndrome72,129. The main differential Similarly, most patients with acrodysostosis and a muta- diagnosis of ectopic bone formation is FOP. Severe vita- tion in PRKAR1A or PDE4D are born SGA5,13,15. Most min D deficiency or hypomagnesaemia might mimic patients born SGA who have PHP or related disorders PTH resistance130,131. A correct diagnosis can have remain short during their adult life (with a mean final extremely important implications for management, as height in PPHP, AHO, PHP1A and acrodysostosis it enables early screening and treatment of endocrine <−2 s.d.)13,15,23,86. By contrast, patients with methylation complications such as PTH and TSH resistance, pre- changes at the GNAS locus display moderately increased vention and management of obesity and short stature, birthweight and birth length (A.L., H.J., A.H.S., G.M. management of ossifications and accurate genetic and and A.R., unpublished observations). Their final adult prenatal counselling. height is within the normal range107. Although they are unspecific features, overweight Evolution of PHP classification and obesity are associated with specific PHP-related The first classification system distinguished PHP vari- disorders, such as PHP1A, PHP1C and acrodysosto- ants as PHP1A, PHP1B, PHP1C and PPHP1,2,27,29,69,132,133. sis5,6,15,23,28,39,62,64. Patients with PHP1A and PHP1B can The subtype assignment is based on the presence or develop early-onset obesity, usually in the first 2 years absence of AHO together with characterization of 28,44,49,108–111 of life . Noticeably, obesity in adulthood is less hormone resistance and determination of Gsα pro- severe and less common than in childhood28. Patients tein activity using in vitro assays132,133. In addition, this with PHP1A might also experience reduced insulin classification excludes phenocopies of PHP, such as sensitivity112,113. acrodysostosis and POH. Since the diseases were molec- Cognitive impairment is commonly reported in ularly characterized, it has become evident that clinical patients with PHP or related disorders; however, its phenotypes often fail to differentiate between PHP

Table 2 | differential diagnoses of PHP and related disorders based on the main clinical presentation leading symptom differential diagnosis Associated signs or comments Hypocalcaemia with Vitamin D deficiency or resistance57,130 • Improvement upon vitamin D therapy elevated PTH • Rickets and alopecia also seen Rickets268 Enlargement of the metaphyses, leg bowing and elevated ALP Hypoparathyroidism due to a mutation in the PTH gene269 Use different assays to confirm the elevated PTH Brachydactyly Tricho–rhino–phalangeal syndrome due to TRPS1 • Dysmorphism: slowly growing and sparse scalp hair, mutations259 laterally sparse eyebrows, bulbous tip of the nose, long flat philtrum, thin upper vermillion border and protruding ears • Hip dysplasia, small feet and a short hallux, exostosis and ivory epiphyses Isolated brachydactyly type E due to HOXD13 • Syndactyly , long distal phalanges and shortening of the mutations270 distal phalanx of the thumb • Hypoplasia or aplasia, lateral phalangeal duplication and/ or clinodactyly Brachydactyly mental retardation syndrome due to Obesity , short stature, brachydactyly and psychomotor and 2q37 microdeletions271 cognitive alterations Turner syndrome due to partial or complete loss of one Short stature, low birthweight, gonadal failure and variable X chromosome272 neurocognitive defects; brachydactyly and Madelung deformity Brachydactyly type E with short stature due to PTHLH Short stature of variable severity and impaired breast mutations75,273 development; oligodontia, delayed tooth eruption and dental malposition; pseudoepiphyses and brachydactyly Ossifications (subcutaneous) Acne vulgaris80 Superficial nodules but no ossification at pathology Cutaneous tumours, primarily pilomatricomas, • No ossification at pathology chondroid syringomas and basal cell carcinomas, and • For secondary osteoma cutis: history of trauma and burn pilar cysts and nevi80; secondary or traumatic osteoma cutis and miliary osteoma Inflammatory conditions such as scars, chronic venous No ossification at pathology stasis, morphea, scleroderma, dermatomyositis and myositis ossificans progressiva80 Ossifications (progressive) FOP due to a recurrent activating missense mutation of Progressive ossification of skeletal muscle, tendons, fascia ACVR1 (ref.10) and ligaments; upper back and neck are the first parts of the skeleton to be affected; trauma alters the natural progression of the disease; congenital malformation of the great toes Tumoural calcinosis due to FGF23 or GALNT3 Deposition of calcium within the skin and/or muscles and mutations274 hyperphosphataemia Early-onset obesity Beckwith–Wiedemann syndrome104,210 Hemihypertrophy and macroglossia Genetic, cytogenetic or syndromic anomalies Progression of obesity through childhood; possible associated with early-onset obesity , including associated features, such as red hair and hypoadrenalism Prader–Willi syndrome and monogenic obesity (mutations in POMC, MC4R, leptin and the leptin receptor)46,275 Early-onset hypothyroidism Congenital hypothyroidism of any cause46,47 Small thyroid and TSH moderately elevated; no other associated features TSH resistance due to mutations in the TSH receptor276 Small thyroid and TSH moderately elevated; no other associated features Hypertension Autosomal dominant hypertension and brachydactyly Short stature type E syndrome239 The list of differential diagnoses is not exhaustive but mentions the main diseases that overlap with pseudo hypopar athyroi dism (PHP) and related disorders. ALP, alkaline phosphatase; FOP, fibrodysplasia ossificans progressive; PTH, parathyroid hormone.

subtypes43,103,134,135. The overlap between the diseases Recommendations (Table 1 and Supplementary Table 1) renders the diagno- 1.13. Consider genetic diagnosis in patients who present sis complex and the classification inadaptable and obso- with one or more major criteria suggestive of PHP lete. In addition, the tools to investigate patients have and related disorders (A+++).

evolved, measurement of Gsα activity in cell membranes 1.14. The classification of PHP and related disorders is not readily available and molecular genetic diagnosis should be amended to include the following (A++): has become the gold standard by which PHP variants • A common pathophysiological framework are distinguished17. • A molecular genetic classification

Molecular diagnosis and three maternally methylated-DMRs (GNAS- A positive molecular test provides important confirma- AS1:TSS-DMR, GNAS-XL:Ex1-DMR and GNAS tion of the clinical diagnosis and allows the categoriza- A/B:TSS-DMR, according to the current nomencla- tion of a patient into a specific subtype of PHP, which ture156). Loss of methylation at GNAS A/B:TSS-DMR is can guide management. detected in all patients with PHP1B45,47,86–88,101,143,151–154. Annotation of each variant (including the interpre- Of the PHP1B cases, 15–20% are familial, with an tation of the variant according to international guide- autosomal dominant mode of inheritance (AD-PHP1B) lines136) and its association with the clinical findings through the maternal lineage17. In this familial form, the within the curated Leiden Open Variation Database methylation defect is usually limited to loss of methy (LOVD) could improve our knowledge of the correlation lation at GNAS A/B:TSS-DMR, secondary to a 3 kb between molecular defects and phenotype. microdeletion on the maternal allele of cis-acting control elements within STX16 (ref.157). Other maternally inher- Molecular confirmation ited deletions and duplications have also been identified GNAS locus. As previously mentioned, the main subtypes in some rare familial cases affecting either an isolated of PHP are caused by de novo or autosomal dominantly GNAS A/B:TSS-DMR 66,158–160 or all four DMRs50,159,161–163 inherited inactivating genetic pathogenic variants or epi- (Supplementary Fig. 1c). genetic alterations (sporadic or genetic-based) within or In nearly all sporadic cases of PHP1B, two or more upstream of the GNAS locus. This region gives rise to DMRs, in addition to GNAS A/B:TSS-DMR, are also multiple non-coding and coding transcripts, including affected, for which no underlying genetic mecha- 154 those encoding Gsα (Supplementary Fig. 1a). GNAS, the nism has been identified (Supplementary Fig. 1d). 17,102 gene that encodes Gsα, is an imprinted gene. It shows In around 8–10% of these sporadic cases, the meth- biallelic expression in most studied tissues, whereas ylation defects are caused by paternal uniparental iso- primarily maternal expression is observed in some disomy of the chromosomal region comprising GNAS tissues (thyroid, renal proximal tubule, pituitary and (UPD(20q)pat)164–168 (Supplementary Fig. 1e). ovary)52,137–140. This tissue-specific monoallelic expres- To date, very few cohorts of patients with PHP1B have

sion of Gsα explains most of the clinical outcomes that been extensively screened for multilocus imprinting dis- depend on the parental origin of the GNAS mutation. turbance (MLID). The incidence of MLID associated 169 92 For instance, because the paternal Gsα allele is mostly with PHP1B ranges from absent to 38% , with most silenced in the renal proximal tubule, GNAS mutations studies quoting a frequency in between these values.

on this allele do not impair Gsα activity. By contrast, if This variation can be partially explained by the small a GNAS mutation is inherited maternally or if it occurs number of patients investigated in each study, the low

de novo on the maternal allele, then Gsα levels and/or sensitivity of molecular techniques or the total number activity are drastically reduced, leading to PTH resist- of imprinted loci assessed. Consistent with most stud- ance in the renal proximal tubule. The molecular and ies of MLID, patients with PHP1B-MLID do not show genetic mechanisms underlying GNAS-related disorders evidence of phenotype differences, and robust meth- are reviewed further in multiple other articles33,141,142. ylation changes are restricted to those with a sporadic In brief, PHP1A is caused by inactivating variants on cause89,92,104,170,171. Despite the important advances made the maternal allele of the GNAS gene within exons 1–13 with high-density methylation array screening, bio (referring sequences NG_016194.1/NM_001077488.1 informatic standardization is required to ensure accurate and LRG_1051), including both point mutations and comparisons between cohorts and accurate description rare gene rearrangements16,23,27,41,45,86,88,97,112,122,143–148 of methylation disturbance in individual patients. (Supplementary Fig. 1b). Mutations can be either mater- GNAS methylation defects can be detected through nally inherited or de novo, with both types of mutation the use of several methods. Methylation-sensitive MLPA having similar incidences17. When mutations affect the (MS-MLPA) enables interrogation of multiple regions paternal allele, they mainly cause PPHP but can also be in a single reaction. A kit from MRC-Holland is avail- responsible for osteoma cutis or POH23,41,83,86,122,145,147,149. able (MS-MLPA ME031 GNAS) to determine the level Point mutations can be easily detected by sequencing of methylation at multiple GNAS sites and to screen for (either Sanger sequencing or next-generation sequenc- STX16 and NESP/AS deletions and deletions encom ing (NGS); particular attention should be given to exon 1 passing GNAS172. Alternative techniques used for the in the latter method, as this exon is particularly CG-rich detection of methylation defects, but that cannot dis and coverage might be incomplete150), whereas genomic criminate epigenetic abnormalities as a result of GNAS rearrangements can be analysed by quantitative meth- deletions, include combined bisulfite restriction analysis ods, such as multiplex ligation-dependent probe ampli- (COBRA), pyrosequencing, methylation-sensitive fication (MLPA) or comparative genomic hybridization single nucleotide primer extension (MS-SNuPE) arrays (aCGH)144. and EpiTYPER155,157,169. By contrast, patients with PHP1B show abnormal pat- When testing for disomy, the identification of the two terns of methylation in the DMRs associated with the (identical) replica copies of a single homologue of a pater- GNAS complex locus45,47,86–88,101,143,151–154. A methylation nal 20q chromosome can be analysed either by micro- defect can be classified as partial or complete and can satellite or short tandem repeat (STR) typing (analysis affect one or multiple DMRs within 20q13 (ref.155). GNAS of the trio, that is, parents and the index case, might be shows differential methylation at four distinct DMRs: one essential for definitive conclusions) or by performing a paternally methylated-DMR (GNAS-NESP:TSS-DMR) single-nucleotide polymorphism (SNP) array164,165,167.

On the basis of the different molecular alterations, 2.10. After exclusion of alterations in genes associated molecular testing must be able to robustly and accu- with PHP and related disorders, the patient should rately detect point mutations, genetic or genomic be referred to an expert centre (A++). rearrangements and methylation defects. Genetic counselling PRKAR1A and PDE4D. Only heterozygous point muta- Despite the clinical and molecular overlap between tions in PRKAR1A (referring sequence NC_000017.11/ PHP and related disorders (Table 1 and Supplementary NM_002734.4) or PDE4D (referring sequence Table 1), once the molecular defect is identified, genetic NG_027957.1/NM_001165899) have been associated counselling is available for patients and families in most with acrodysostosis5,6,13–15,20,62–64. expert centres. Other genetic or genomic alterations have been No correlation exists between the severity of identified in these genes, but they are not associated the parental phenotype and that of the affected off- with PHP and related disorders. For example, hetero spring, as interfamilial and intrafamilial variability zygous genomic rearrangements (deletions and duplica- has been observed for the same mutation, and the tions) at 5q12.1 encompassing PDE4D are associated imprinting effect at the GNAS gene also affects clinical with a novel intellectual disability syndrome (OMIM outcomes16,64. #615668) without acrodysostosis and with a specific When dealing with GNAS gene defects, the parental phenotype that diverges from that of patients with point origin of the mutation and clinical heterogeneity make mutations in PDE4D14. Furthermore, mutations and genetic counselling a challenging task, mainly owing to deletions in 17q24.2-q24.3 encompassing PRKAR1A four factors. First, paternal inheritance (or a mutation have been associated with Carney complex173, which in the paternal allele) can lead to either a mild expres- results from activation of protein kinase A signalling174. sion of PPHP or a severe expression of POH. Why paternal inheritance of a GNAS mutation should result Recommendations in PPHP in some families and POH in others remains 2.1. Molecular diagnosis of individuals with a suspected unclear10,74,84. Second, when the mutation involves the diagnosis of PHP must include DNA sequence, maternal allele, it will lead to PHP1A or PHP1C. Third, methylation and copy number variant (CNV) patients have a 50% chance of transmitting the molecu analyses at the GNAS locus (Fig. 2) (A++). lar defect, and depending on their sex, the descendant 2.2. When the clinical pattern is highly suggestive of will develop PPHP or POH (when the patient is male) or alteration of a specific gene, Sanger sequencing of PHP1A or PHP1C (when the patient is female). Fourth, that gene is proposed (A++). there is no clear correlation between the type or location 2.3. When the clinical presentation is not suggestive of of GNAS mutations and the disease onset, severity of a specific gene, a targeted gene panel encompassing endocrine resistance, neurocognitive phenotype or the genes that encode proteins involved in the PTH– number of AHO features. parathyroid hormone-related protein (PTHrP) sig- When methylation alterations at the GNAS locus nalling pathway might be performed. If gene panel are present, the recurrence risk depends on the under sequencing is used, it should fulfil the recommended lying genetic defect, if present, and the parental origin. guidelines175 for design and reporting (A++). Paternally inherited STX16 or NESP/AS deletions are not 2.4. If a sequence variant is identified in any gene associ- associated with methylation defects, so that the descend- ated with PHP and related disorders, its pathogenic- ants of a male carrying any of these deletions have a 50% ity should be assessed according to the established chance of inheriting the genetic defect; however, if they standards and guidelines136 (A+++). inherit the genetic defect, their GNAS methylation status 2.5. The parental origin of the variant could have impor- will be normal, and they will not develop PHP1B157. If a tant clinical implications; thus, parental testing female carries the STX16 or NESP/AS deletion, each of is indicated when a genetic alteration is detected her descendants has a 50% risk of inheriting the genetic (A++). defect, and if the descendant inherits the defect, he or she 2.6. If the variant is de novo, the allelic origin might have will present with a methylation defect affecting only GNAS to be determined (B+++). A/B:TSS-DMR or the complete GNAS locus (depending 2.7. In certain cases, such as UPD(20q)pat or gross dele- on the inherited deletion)18. In both cases, the descendant tions, additional genetic studies might be advisable will develop PHP1B45,47,86–88,101,143,151–154. Although translo- (A++). cations of chromosome 20 resulting in UPD(20)pat are 2.8. Technical limitations must be taken into consider- very rare, parental karyotyping is recommended to estab- ation when reporting negative results and included lish the risk of recurrence. If no genetic alteration can be in the genetic report. For instance, mosaicism can identified that is associated with the methylation defect, be considered if a negative result is observed in a the recurrence and transmission risks are expected to be patient with high clinical suspicion through analysis similar to that of the general population. of tissues other than blood (for example, saliva or a Pathogenic variants at PRKAR1A and PDE4D mostly buccal swab) or the use of alternative techniques, occur de novo5,6,13–15,20,62–64. According to the pattern of such as deep sequencing (A++). autosomal dominant inheritance, patients have a 50% 2.9. For each de novo pathogenic variant, or variants of of chance of passing on the molecular defect and the unknown significance, parental germline mosaicism disease to their descendants, independently of the sex should be considered (A++). of either the progenitor or the descendant.

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%CPFKFCVG &GNGVGTKQWUOWVCVKQPU 9'5QT9)5 IGPGUETGGPKPI CV56:QT0'52! Fig. 2 | Molecular algorithm for the confirmation of diagnosis of PHP and related disorders. If patients present with Albright hereditary osteodystrophy (AHO), genetic alterations at GNAS should be studied, including point mutations (sequencing) and genomic rearrangements (such as multiplex ligation-dependent probe amplification (MLPA) and comparative genomic hybridization arrays (aCGH)). Once the variant is found, its pathogenicity should be confirmed according to guidelines136, and, when possible, the parental origin should be determined. In the absence of AHO, epigenetic alterations should be analysed first. According to the results obtained for the methylation status, further tests are needed to reach the final diagnosis: if the methylation defect is restricted to transcription start site (TSS)–differentially methylated region (DMR) at exon A/B of GNAS (GNAS A/B:TSS-DMR), STX16 deletions should be screened for, and, if present, the diagnosis of autosomal dominant- pseudohy popar athyroidism type 1B (AD-PHP1B) is confirmed; if the methylation is modified at the four DMRs, paternal uniparental disomy of chromosome 20 (UPD(20q)pat) should be screened for ; in absence of UPD(20q)pat, deletions at NESP should be screened for ; if no genetic cause is identified as the cause of the methylation defect, the sporadic form of the disease (sporPHP1B) is suspected. After exclusion of the GNAS locus as the cause of the phenotype, and in patients with AHO, pseudo hypopar athyroidism (PHP)-related genes (that is, at least PDE4D and PRKAR1A) should be sequenced. Squares in light red indicate the technology ; blue, the final molecular confirmation; red, no molecular alteration; and grey , future or research steps are suggested. ICRs, imprinting control regions; MLID, multilocus imprinting disturbance; NGS, next-generation sequencing; PHP1A , pseudohypoparathyroidism type 1A; PHP1B, pseudohypoparathyroidism type 1B; POH, progressive osseous heteroplasia; PPHP, pseudopseudohy popar athyroi dism; RT-PCR , reverse-transcription PCR; SNP, single-nucleotide polymorphism; STRs, short tandem repeats (microsatellites); UPD, uniparental disomy; VUS, variant of unknown significance; WES, whole-exome sequencing; WGS, whole-genome sequencing.

The identification of a pathogenic variant in an Long-term treatment of hypocalcaemia associated index case enables a correct diagnosis and the possi- with PTH resistance is similar but usually more aggres- bility of predictive genetic testing in relatives, which sive than that of primary hypoparathyroidism, with can be excluded from further follow-up if the result is the use of active vitamin D metabolites (calcitriol) or negative176. In parents of an index case, it is essential to analogues (alfacalcidol) and oral calcium supplements exclude mosaicism116,177,178 or to indicate this possibility as and when required179. The current approach is to at the counselling session. reduce the serum level of PTH to the upper portion of the reference range to avoid suppression of PTH, which Recommendations can be associated with hypercalciuria and renal calcifi- 2.11. Genetic counselling should be adapted to the cation. Serum levels of PTH that are at the upper limits different molecular findings for the GNAS locus of the reference range are sufficient to enhance calcium (B++) and the PRKAR1A and PDE4D genes (A++) reabsorption in the distal renal tubule, thus helping to according to Table 3. prevent hypercalciuria34. However, PTH levels should not be too high, as long-standing PTH excess might Prenatal testing have adverse effects on skeletal mineralization or on There are very few cases with antenatal diagnosis of the growth plate71,180–182. Accordingly, treatment with PHP or PPHP, as their clinical manifestations (including oral calcium and active vitamin D supplementation can growth retardation, subcutaneous ossifications and TSH be used to target a higher serum level of calcium than resistance) are usually not specific within this period. in patients with PTH-deficient hypocalcaemia, where The bone dysplasia observed in patients with acrodysos- treatment targets the low–normal or slightly reduced tosis might display a prenatal onset, mostly when caused range of calcium. As a result of the preserved sensitivity by PRKAR1A mutations6. of the distal convoluted tubules, patients with PHP and related disorders very rarely develop hypercalciuria34. Recommendations However, several experts involved in this Consensus 2.12. Even if all the molecular defects associated with PHP Statement have encountered episodes of nephrolithiasis and related disorders were known and technically in patients with PHP1A and PHP1B, particularly after available for testing in preimplantation or prena- completion of pubertal growth, when the doses of oral tal diagnosis, ethical and legal concerns regarding calcium and active vitamin D analogues can be reduced the pertinence of performing these tests should be (G.M., P.K, A.R. and A.L., unpublished observations). taken into consideration, with appropriate genetic Chronic hypocalcaemia and associated hyperphos- counselling (A++). phataemia can result in intracranial deposition of cal- 2.13. If prenatal testing is considered, methylation cium, a feature usually referred to as Fahr syndrome65. analysis is not recommended (A++). These calcifications occur predominantly in the basal ganglia but might extend widely to the thalami and the Management cortex. This form of ectopic calcification is due to ele- To date, no prospective clinical trials have been con- vated levels of the calcium–phosphorus product and ducted in patients with PHP and related disorders that hence has not been described in patients with PPHP focus on the management and outcomes of treatment in or POH or those with a mutation in the PRKAR1A or these disorders. Large cohorts of molecularly diagnosed PDE4D genes5,6,8,62,183,184. patients are needed for recruitment into clinical stud- Other ectopic depositions of calcium and phospho- ies, which should also include long-term follow-up. As rus occur in the eyes, which lead to cataracts (peripheral a result of the rarity of the disease, cohorts and research lenticular opacities). Corneal opacities, macular degen- forces are scattered; there is a need to coordinate and eration, nystagmus, anisocoria, papilloedema, tortuos- implement multicentre international clinical trials. ity of retinal vessels and microphthalmia have also been A multidisciplinary follow-up and early, specific reported8,185–188. interventions are necessary for efficient therapeutic Long-term and excessive secondary hyperparathy- management of these patients. Table 4, which is based roidism can result in osteitis fibrosa cystica and slipped on the literature analysis and authors’ expertise, summa- capital femoral epiphysis, which might require a com- rizes the main interventions that should take place during bination of medical and surgical management189,190. the follow-up of patients with PHP and related disorders Patients with GNAS mutations involving the paternal and gives a suggested frequency for the interventions. allele or PDE4D mutations usually do not develop PTH resistance over time15,20,23,60,62,63. Recommendation Additional complications of PTH resistance include 3.1. The diagnosis and management of patients with dental manifestations, such as failure of tooth eruption, PHP and related disorders should be performed by short blunted roots, dental pulp alterations, hypodon- a multidisciplinary team of specialists (A++). tia and enamel hypoplasia. Finally, increased uric acid excretion has been reported in two families with PHP1B, Management of PTH resistance which suggests a possible role for PTH resistance in the Even though PTH resistance is the hallmark of PHP and renal handling of this analyte191,192. Increased excretion is referenced in the name of the disorder, very few stud- of uric acid and phosphorus might increase the risk of ies have focused on the natural history of its appearance, developing kidney stones if urinary levels of calcium and almost none on its management. are increased. These last complications, when present,

Table 3 | guidance for genetic counselling in PHP and related disorders according to each molecular defect locus Molecular Molecular defect Affected cases Theoretical clinical predictive Further tests diagnosis allele (n or %) recurrence risk outcome (when inherited) GNASa Genetic defect Heterozygous loss- Mat 35.9% 50% Affected pt PHP1A Maternal testing of-function mutation for carrier status Pat 5.9% 50% Affected pt PPHP and/ Paternal testing or POH for carrier status Partial or total locus Mat 1.8% 50% Affected pt PHP1A Maternal testing deletion or inversion for carrier status Pat 0.2% 50% Affected pt PPHP and/ Paternal testing or POH for carrier status Sporadic Broad LOI (all GNAS Mat 38% ND Affected pt sporPHP1B Research methylation defect DMRs) Isolated LOM GNAS Pat 2% ND Affected pt sporPHP1B Research A/B:TSS-DMR Uniparental disomy – 2.7% Low; high when Affected pt sporPHP1B Chromosome (UPD(20q)pat) a translocation is analysis of present proband and father Inherited Isolated LOM GNAS Mat 13.5% 50% Affected pt AD-PHP1B Maternal testing methylation defect A/B:TSS-DMR + STX16 for carrier status ICR deletion STX16 ICR deletion Pat ND 50% Unaffected carrier Not required Broad LOI + NESP/AS Mat ND 50% Affected pt AD-PHP1B Maternal testing ICR deletion for carrier status NESP/AS ICR deletion Pat ND 50% Unaffected carrier Not required PRKAR1Ab Genetic defect Heterozygous mutation Not relevant 79 50% Affected pt Parental testing for carrier status PDE4Db Genetic defect Heterozygous mutation Not relevant 43 50% Affected pt Parental testing for carrier status AD-PHP1B, autosomal dominant pseudo hypopar athyroi dism type 1B; DMR , differentially methylated region; ICR , imprinting control region; LOI, loss of imprinting; LOM, loss of methylation; Mat, maternal; ND, not determined; Pat, paternal; PHP, pseudohypothyroidism; PHP1A , pseudohy popar athyroidism type 1A ; POH, progressive osseous heteroplasia; PPHP, pseudopseudohypoparathyroidism; pt, patient; sporPHP1B, sporadic pseudohypoparathyroidism type 1B. aPercentages obtained from Elli et al.17 bNumbers extracted from the corresponding Leiden Open Variation Database (LOVD) web referred to total carriers with public variants (it varies with time).

should be managed according to good clinical practice. to general guidelines for the management of acute No specific recommendations can be formulated at the hypocalcaemia in hypoparathyroidism179. These moment given the rarity of their presentation. patients should also be concomitantly treated with active vitamin D metabolites or analogues (A+++). Recommendations 3.4. In patients with substantial and progressive increases 3.2. At diagnosis or before initiation of treatment, in levels of PTH and hyperphosphataemia, treatment we recommend the monitoring of serum levels with active vitamin D metabolites or analogues of PTH, calcium, phosphorus and calcifediol. could be considered, independently of the presence Measurement of PTH, calcium and phosphorus of hypocalcaemia. Calcium supplements should be should be performed regularly (every 6 months considered, depending on the dietary calcium intake. in children and at least yearly in adults) with the Serum levels of phosphorus should be monitored exception of patients carrying either a GNAS muta- during treatment with vitamin D metabolites or tion on the paternal allele or a PDE4D mutation in analogues and calcium supplements (A++). whom, apart from at diagnosis, routine assessment 3.4.b. Treatment with active vitamin D metabolites or is not necessary. Monitoring of serum levels of cal- analogues could also be considered when lev- cium should be more frequent in symptomatic els of PTH are more than twice the upper level individuals, during acute phases of growth, during of normal, independently of the presence of acute illness and during pregnancy and breastfeed- hypocalcaemia (B+). ing, when dose requirements for active vitamin D 3.5. The objectives of conventional management of metabolites or analogues might change. Calcifediol PTH resistance include maintenance of serum lev- levels should be normalized and maintained in the els of calcium and phosphorus within the normal normal range in all patients (A+++). range while avoiding hypercalciuria (age and size 3.3. Severe symptomatic hypocalcaemia requires corrected) and lowering PTH levels as permitted immediate correction, which might include intra- by serum and urinary levels of calcium. We recom- venous administration of calcium salts according mend the use of the active vitamin D metabolite

Table 4 | Summary of the main interventions during the follow-up of patients with PHP and related disorders Action points infancy (newborn Early childhood late childhood to Adulthood to 2 years) (2–6 years) adolescence Anticipatory guidance Family support ✓ ✓ ✓ NA Genetic counselling At diagnosis At diagnosis At diagnosis At diagnosis Medical evaluation Linear growth ✓ ✓ ✓ NA Weight gain and BMI ✓ ✓ ✓ ✓ Descended testis ✓ ✓ If not checked before If not checked before Blood pressure NA ✓a ✓ ✓ Development and/or ✓ ✓ S S cognition Psychosocial evaluation NA ✓ S S Ectopic ossifications ✓ ✓ ✓ S Orthodontic and/or dental NA ✓ ✓ S Bone age radiography NA ✓ (in case of growth ✓ (in case of growth NA deceleration) deceleration) Calcium–phosphorus ✓ ✓ ✓ ✓ metabolism Age-appropriate renal ✓b ✓b ✓ ✓b imaging Thyroid ✓ ✓ ✓ ✓ Puberty NA NA ✓ (biochemistry in NA case of retardation) GH secretion NA ✓ ✓ S Glucose and lipid metabolism NA ✓ ✓ ✓ Fertility NA NA S S GH, growth hormone; NA , not applicable; PHP, pseudohypopar athyroi dism; S, subjective (by history and physical examination); ✓, to be performed at diagnosis and annually thereafter. aAt least once per year, with an appropriate sized cuff. bAnnually in case of increased excretion of urinary calcium or nephrocalcinosis.

calcitriol or the active vitamin D analogue (alfa- 3.9. Phosphate binders (other than calcium) are rarely, calcidol) with or without calcium supplementation if ever, indicated in the management of severe and as the mainstay of treatment of chronic hypo long-term persistent hyperphosphataemia (B++). calcaemia. Patients should not be treated with 3.10. We recommend regular dental reviews every PTH or PTH analogues. During treatment, levels 6–12 months during childhood and as clinically of PTH, calcium and phosphorus should be mon- indicated in adults (A++). itored every 6 months in asymptomatic patients and more frequently when clinically indicated. Management of TSH resistance Patients and/or their family should be instructed In patients with PHP1A, the average level of TSH is about symptoms and signs of hypocalcaemia and 14.1 ± 10.3 mUI/l, with a range of 1.4 mUI/l to 46.0 mUI/l hypercalcaemia (A++). (refs24,30,38,67,98,100,117,119,122,123,129,144–146,149,193–205) . 3.6. We recommend appropriate renal imaging to A prompt diagnosis of hypothyroidism after birth and evaluate nephrocalcinosis at transition (B+). initiation of treatment does not seem to prevent the 3.7. We recommend age-appropriate179 renal imaging development of motor or cognitive delay24. to monitor for the development or worsening of In patients with PHP1B, the degree of TSH resistance nephrocalcinosis in patients with persistent hyper- can vary with time52,53,102,206. The average level of TSH calciuria on repeated measurements and as clini- is 5.3 ± 4.7 mUI/l (4.8 ± 3.4 mUI/l and 5.4 ± 5.2 mUI/l cally indicated (B++). in autosomal dominant PHP1B and sporadic PHP1B, 3.8. For the evaluation of long-term consequences of respectively), ranging from 0.8 mUI/l to 50.0 mUI/l hypocalcaemia and hyperphosphataemia, a brain (refs36,44,46–49,51,53,54,87,89,91,102,105,131,143,153,164,166,169,178,200,206–210). CT scan is indicated only when neurological man- The literature does not contain specific data on how ifestations are present. Ophthalmological exami to treat TSH resistance in patients with PHP and related nation is recommended to diagnose or exclude disorders. Indications for treatment, drug formulations,

cataracts (A++). doses and target serum concentrations of TSH and T4

should be the same as those used in any other form of growth advantage compared with her sister who did hypothyroidism or subclinical hypothyroidism. not receive the analogues217. Data from a clinical trial In general, patients should be screened for auto- (NCT00209235)220 on final height and on the association immune thyroid disease. Nevertheless, because autoimmune of puberty blockers are pending. thyroid disease is highly prevalent, the presence of auto Finally, in the context of prenatal growth restric- immunity will not rule out concomitant TSH resistance tion, even if the GH secretion is deficient, the authors’ in a given patient (see recommendation 3.11). experience suggests using doses of rhGH higher than those usually given in children with idiopathic GH defi- Recommendations ciency, for instance, the doses used in patients with SGA 3.11. Evaluation of thyroid function (including autoanti- (L.d.S., A.L., G.M. and A.R., unpublished observations). bodies in adolescents and adults) for early detection Overall, additional published data are required before a of TSH resistance and early intervention is recom- recommendation on the use of GH therapy, or any other mended in all patients with PHP and related dis- growth modifying treatment, in this group of patients orders at diagnosis. Thereafter, TSH monitoring is can be made. recommended every 6 months in patients <5 years of age and yearly in older children and adults (A++). Recommendations 3.12. The indication or indications to treat hypothyroid- 3.13. We recommend careful monitoring of height in ism, the dosage of levothyroxine and the therapeu- children at every control examination (at least tic goals should be the same as for any patient with every 6 months) until final height is reached hypothyroidism or subclinical hypothyroidism211–213 (A+++). (A++). 3.14. We recommend monitoring of skeletal maturation using plain radiography in all children (with the Management of growth and GH deficiency ex c e p tion of th o se wi t h PH P 1B) an d ev a lu a t ion With the exception of PHP1B, a large proportion for GH deficiency in all children in the context (around 80% of patients with PHP1A, 50–70% of of statural growth deceleration. As most patients patients with PPHP and almost all patients with develop GH deficiency due to GHRH resistance, acrodysostosis) of patients with PHP and related clinical and/or biochemical evaluation of the GH– disorders have adult short stature5,13–15 ,23 , 24 ,28 , 39 , insulin-like growth factor 1 (IGF1) axis should be 45,53,54,62–64,86,89,97,104,105,107,113,122,143,145,146,153,205,206,214–216. Overall, performed in all patients, typically around the age the final height deficit in patients with PHP1A and PPHP of 3–6 years and repeated as necessary (B++). is severe, approximately −2.5 s.d., with mean heights of 3.15. Patients with GH deficiency should be treated with 149 cm in women and 155 cm in men28,122,217. In patients rhGH. Data are needed on the outcomes of GH treat- with PHP1B, despite a great variability in adult height, ment in children who are not GH deficient before mean stature is not significantly different from that of recommending this treatment in these patients as the general population107. Patients with acrodysostosis well (A++). have adult heights that are even shorter than those of 3.16. Adults with GH deficiency might be considered patients with PHP1A, with a mean height of −3.5 s.d. for treatment with rhGH; however, specific proof (−8.8 to −0.5)5,14,15,62–64. The height deficit in patients with of benefit in this population is lacking, and treat- PHP develops over time. Small cohorts of patients with ment should be given according to country-specific PHP1A have shown declining growth velocity a few years regulations (B++). after birth, a lack of a pubertal growth spurt and prema- 3.17. Patients with PHP who are born SGA who do not ture cessation of statural growth86,97,107,217. This finding is exhibit appropriate catch-up growth might qualify also supported by observations from the authors’ day- for treatment with rhGH; however, caution should to-day practice (L.d.S., H.J., A.L., P.K., G.M., G.A.M.-M., be exercised in patients with ectopic ossifications, as O.M., A.R. and Su.T., unpublished observations). no data are available on the possible effect of rhGH Bone age is advanced by more than 2.0 s.d. treatment on the evolution of ossifications (B++). beyond chronological age in 70–80% of patients with PHP1A97,215,217,218. Advanced growth plate maturation is Alterations in gonadal function consistently described in patients with acrodysostosis5,15,63. Gonadal function and puberty. The pubertal growth spurt The severity of the dysostosis reduces the reliability of might be blunted or absent in both girls and boys with using bone age determinations to predict adult height219. PHP1A. Although the basis for this defect is unknown, it We know from our practice that phalanx epiphyses might could relate to insufficient sex steroid production or pre- close as early as 3 years of age in these children (A.L., mature epiphyseal closure120. Systematic data are scarcer G.A.M.-M., A.R. and Su.T., unpublished observations). for PHP1B and PPHP than for PHP1A, but these patients Most patients with PHP1A (50–80%) develop GHRH are thought to have normal gonadal function206. Similarly resistance and consequently GH deficiency45,86,97,143,146,20 to PHP1A, variable resistance to gonadotropins has been 6,215,218. Recombinant human growth hormone (rhGH) described in patients with acrodysostosis and mutations treatment increased growth velocity in one study con- in the PRKAR1A gene15, as well as in anecdotal reports of ducted in eight prepubertal children with PHP1A and single patients with mutations in PDE4D14. Furthermore, GH deficiency. It is of interest that in this study, one despite basal gonadotropin and sex steroid levels that female patient whose oestrogen production was blocked are within normal limits, menstrual irregularities seem by GnRH analogues did have evidence of a long-term to be common among female patients with PHP1A120.

According to the experts’ experience, cryptorchidism is 3.23. In patients with PHP and related disorders, natu- highly prevalent in patients with PHP1A, leading to the ral and induced pregnancies should be monitored hypothesis of a possible role of hormone resistance in the from an obstetrical point of view in the same way pathogenesis of PHP1A (A.L., P.K., Su.T., A.R. and G.M., as any other pregnancy. However, dosages of active unpublished observations). forms of vitamin D and levothyroxine might have to be adjusted. The possibility of vaginal delivery Fertility and pregnancy. Unassisted and unevent- might be limited as a result of reduced pelvic size ful pregnancies have been reported in female and decreased range of motion of the hips due to patients with PHP1A120,221 and autosomal dominant local ossifications (A+). PHP1B44,72,153,160,216; these pregnancies are more often seen 3.24. Management of hypocalcaemia and hypothyroidism in women with PPHP, who give birth to offspring with should follow the current guidelines available for PHP1A24,36,45,117,122,221–223. In a few cases, either infertility193 the management of hypoparathyroidism and hypo- or the need for the use of an assisted reproductive tech- thyroidism during pregnancy. The newborn should nique to obtain pregnancy47,224,225 have been reported. No be evaluated for levels of TSH, calcium and phos- data are available on lactation, but there are no specific phorus. Lactation is not contraindicated, but close reports that these patients are unable to lactate. follow-up and clinical monitoring (particularly of weight) of the baby are advised (B++). Menopause. No data are available on menopause and its 3.25. Patients with PHP and related disorders have sev- timing in women with PHP and related disorders. eral potential risk factors for osteoporosis (hypogonadism, chronic elevation of PTH and GH Osteoporosis. The prevalence of osteoporosis in patients deficiency). However, owing to the lack of evidence with PHP and related disorders is unknown. In these of increased fracture risk, there is no indication to patients, bone loss might occur as a result of untreated perform routine dual-energy X-ray absorptiom- hypogonadism, long-term excess levels of PTH, GH etry (DXA) measurements in patients with PHP deficiency and/or the onset of physiological meno- and related disorders. If osteoporosis is diagnosed, pause. The physiological action of PTH on bone is management should take into account, whenever mainly to promote bone resorption, but the extent to possible, treatment of the underlying secondary which PTH signalling in bone is defective in patients cause for bone loss (hypogonadism, postmenopau- with PHP and related disorders is not completely clear. sal status or related to sustained elevation of PTH The bone remodelling response to PTH, which is inde- levels and GH deficiency) (B++). pendent of vitamin D action, seems to be intact in these patients180, which suggests a possible increased risk of Management of other hormone resistances osteoporosis in patients with sustained increased lev- Resistance to additional hormones that mediate their

els of PTH despite treatment with calcium and vitamin actions through Gsα-coupled receptors, as well as pro- D. Nevertheless, bone density seems to be normal to lactin deficiency, has also been previously reported; increased in patients with PHP1A, in particular in those however, the clinical relevance of these abnormalities who do not have excessive circulating levels of PTH218. remains to be established132. Early studies described the presence of resistance to calcitonin114, and this finding was Recommendations subsequently confirmed in case reports and in a small 3.18. Tanner staging of sexual maturation should be case series of patients with PHP1A115. Unpublished but performed at regular intervals to monitor puber- consistent observations from the authors indicate that, tal progression in all patients with PHP or related among the other hormone resistances, hypercalcitoni- disorders (A+++). naemia is present in a substantial subset of patients with 3.19. Testicular descent and location should be PHP1A and PHP1B (L.G., A.L., H.J., G.M., A.R., L.R. and assessed in males with PHP or related disorders. A.H.S., unpublished observations). No published evidence Cryptorchidism, when present, should be cor- indicates that hypercalcitoninaemia leads to clinically rected and managed according to the standard significant C cell hyperplasia or medullary thyroid carci- recommendations (A++). noma in PHP and related disorders. Adrenal resistance to 3.20. We do not recommend routine biochemical adrenocorticotropic hormone (ACTH) and other hor- assessment of the gonadal status unless clinically mone resistances are not documented to occur in patients indicated. Hypogonadism, when present, should with PHP1A, apart from a few old anecdotal reports. be treated with sex hormones following the same standard criteria, doses and follow-up as any other Recommendation form of hypogonadism (B++). 3.26. We do not recommend routine calcitonin measure- 3.21. After puberty, menstrual history should be ment or screening for additional hormone resistances collected at each follow-up visit, and biochemical in patients with PHP and related disorders (A+). evaluation should be requested in the presence of oligomenorrhoea or amenorrhoea in women and the Obesity and other metabolic issues presence of hypogonadal symptoms in men (B+++). Children with PHP1A and obesity show both decreased 3.22. In case of infertility, assisted reproductive treat- resting energy expenditure compared with controls with ment can be considered according to national obesity and hyperphagic symptoms similar to those seen guidelines (A+). in BMI-matched controls with obesity39,111,112. In older

(late infancy, adolescence and young adulthood) patients, glucose intolerance and insulin resistance developed this hyperphagic trait seems to abate111, and energy before the onset of obesity235. expenditure seems to improve to low–normal113. As a As for the lipid profile in PHP and related disorders, consequence, obesity is less pronounced in adulthood in a small series of patients with PHP1A, cholesterol, than in childhood28. triglycerides and/or LDL levels were either elevated or With the exceptions of the case reports of a patient at the upper end of the normal range97. No data are avail- with PHP1C successfully treated with a cannabinoid able regarding lipid profiles in the other PHP subtypes receptor type 1 (CB1) antagonist99 and of a patient with and related disorders. PHP1A treated with a gastric bypass226, there are no Finally, blood pressure and its regulation have been specific reports concerning specialized management of very poorly investigated in PHP and related disorders, obesity in these patients. and the scarce available data date back to the 1980s236. Early-onset obesity is observed not only in patients Nevertheless, hypertension is frequently observed in with PHP1A but also in patients with PHP1B44,46,86,158. The young adults with PHP and related disorders. In 1988, increased weight gain might start within the first months elevated blood pressure was reported in 53% of adult of life in some patients, similar to what is observed in patients with PHP and related disorders, with a similar patients with monogenic defects of the leptin–melano- prevalence in clinically defined patients with PHP1A cortin pathway227,228. This finding highlights the need for and PHP1B237. More recently, the case of a young patient close monitoring of weight in all patients with PHP and with PHP1B who had juvenile renin-dependent hyper- related disorders, not only in those with PHP1A. tension has been reported238. A direct link between the Findings from animal models suggest that the under- cAMP signal transduction pathway and hypertension lying mechanism contributing to obesity is related to the has been demonstrated by the finding of PDE3A muta-

effect of Gsα imprinting in the CNS. In the hypothalamus, tions in families with autosomal dominant hyperten- 229 21,239 Gsα signals through melanocortin and thereby affects sion and brachydactyly type E syndrome , and the energy expenditure. This finding suggests that drugs mod- mechanism underlying hypertension in these patients ifying the melanocortin pathway might be effective for is thought to be related to increased peripheral vascular weight control in patients with PHP and related disorders. resistance due to vasoconstriction. Sleep apnoea is a well-known complication of obesity. Sleep disturbances with daytime somnolence have Recommendations been reported in a series of patients with PHP1A230 and 3.27. We recommend regular monitoring of BMI and were more frequent (4.4-fold higher relative risk) than eating behaviour (Table 4). Educational pro- in participants without PHP1A who had similar levels of grammes, as well as psychological support, should obesity230,231. Irrespective of the underlying mechanism, be provided to patients and families when obesity untreated sleep apnoea is associated with poor memory and/or eating disorders are present and even in the and concentration, increased risk of heart disease and presence of a normal BMI as a preventive strategy, impaired glucose metabolism232. Effective treatment of as these patients are at high risk. Dietary counsel- sleep apnoea might mitigate these risks and even improve ling should take into account that these patients school performance233. In addition to sleep apnoea, other have decreased resting energy expenditure (A++). pulmonary disorders (such as an increased prevalence 3.28. We recommend that all patients with PHP and of asthma) have also been associated with PHP1A related disorders should be evaluated for symptoms (50–75%), PPHP and PHP1B39,46,230,234. such as restless sleep, snoring, inattentiveness and Metabolic consequences of PHP and related disorders daytime somnolence, and, if symptoms are present, have not been characterized. Nevertheless, in addition to polysomnography is recommended (B++). obesity, impairment of glucose metabolism and hyper- 3.29. Lipid and glucose metabolism should be monitored tension are present in a large subset of patients112,113. For on a regular basis (Table 4) (B++). instance, patients with PHP1A have decreased insulin 3.30. Blood pressure should be monitored regularly sensitivity that appears early during childhood, which (Table 4) with an appropriately sized cuff. Patients seems to be only partially related to the degree of obesity. with hypertension should be treated as clinically These data have been published in single case reports109, indicated (B++). as well as in two small series of children and adolescents (aged 2–18 years)112,113, but are also confirmed in daily Management of ectopic ossifications clinical practice (A.L., G.A.M.-M., A.H.S. and A.R., In PHP and related disorders, particularly POH, trig- unpublished observations). gering environmental factors for ectopic ossifications, In a series of ten children with PHP1A, levels of such as trauma, infection and/or metabolic or immune 240 HbA1c and fasting insulin were normal in all partici- abnormalities, have not been systematically studied ; pants but one97. By contrast, in ten adults with PHP1A, however, clinical experience strongly suggests that four already had a diagnosis of type 2 diabetes melli- these factors do not contribute to new ossifications. By

tus, the fasting plasma levels of glucose and HbA1c were contrast, in FOP, another disease featuring ectopic bone higher than the controls with obesity, and they showed formation, external factors, such as trauma or infec- decreased insulin sensitivity113. These findings indicate tions, are well-known triggering events contributing to that factors other than obesity might contribute to lower new bone formation or worsening of existing ones10. insulin sensitivity in these patients and are consistent A systematic literature search led to the following with observations in a mouse model of PHP1A in which observations. First, no external factor has been shown

to induce the development of ectopic ossifications in instructed about self-examination. We recommend patients with PHP1A, PPHP or POH. Second, no cor- documenting the following at each visit: location relation exists between PTH levels or serum levels of and size of ossifications; involvement of joints calcium–phosphorus and the occurrence of ectopic ossi- and impairment of movement and bone growth; fications. Third, ectopic ossifications often occur in loca- predilection of lesions towards areas exposed to tions subjected to high pressure loads, such as the heel81. increased pressure due to weight bearing (feet and Fourth, plate-like or severe ossifications might develop ankles); assessment of triggering events (trauma, within the first year of life and progressively invade the infection, inflammation and surgery); association deep tissues58. Finally, paternally inherited mutations with pubertal development; and treatment with at GNAS, mainly those leading to a truncated protein, rhGH (A++). have been reported as contributing to the development 3.32. Regular imaging of ectopic ossifications is not of ectopic ossifications41,145. indicated (A++). As a result of the rareness of these conditions, limited 3.33. Imaging of ossifications should be performed information is available about prognosis. Currently, no using CT or MRI, depending on localization, effective treatments exist for the management or pre- when the lesions are painful, symptomatic, jeop- vention of ectopic ossifications. Surgical removal of the ardize joint or organ function or are being consid- ectopic ossifications can be performed when the lesions ered for surgical excision (A++). are well delimited. This is rarely the case in patients 3.34. When the diagnosis underlying ectopic ossifica- with POH, but it can be effective in those with osteoma tions is doubtful and when FOP has been excluded, cutis and in superficial lesions associated with PPHP there is no contraindication to perform a biopsy or PHP1A with successful long-term results58,59,81,241. In of the lesion, as no evidence suggests that inflam- most cases, surgical resection is followed by recurrence mation or trauma leads to progression of ectopic or complications3,83,242–245. Successful functional reposi- ossifications in PHP and related disorders (B++). tioning of a joint after the development of a contracture 3.35. Physical therapy and meticulous skin care are the due to ectopic ossifications was reported in one child243. most important approaches for the prevention of Amputations are sometimes needed in the setting of development and/or progression of ectopic ossifi- severe growth retardation and functional ankyloses83 or cations. Surgical excision should be considered in following the development of severe infections in recur- the presence of delimited, superficial lesions asso- rent skin ulcerations242. ciated with pain and/or movement impairment. Bisphosphonates have been proposed to prevent the The patient should be referred to a surgeon with post-surgical complications and recurrences of ossifi- experience in the management of ectopic ossifications owing to their known effect on inhibiting bone cations. In extensive ossifications around a joint, turnover. Although we are lacking data from controlled avoid immobilization (through casts), as this might trials, in five patients with neurogenic or post-traumatic lead to ankyloses of the joint (B+). ectopic ossifications, pamidronate seemed to prevent 3.36. There is no evidence for recommending the use of recurrences79. Pamidronate has also been used in a single nonsteroidal anti-inflammatory drugs, bisphos- patient with POH as a primary treatment of progressing phonates or steroids in primary or peri-surgical heterotopic ossifications, with an apparent delay in new treatment of asymptomatic ectopic ossifications (B+). bone formation, rather than a change in the ectopic bone itself246. Etidronate has also been used with an apparent Brachydactyly and orthopaedic issues beneficial effect on the progression of ectopic ossifica- Skeletal complications. Brachydactyly might contribute tions in one patient with a POH-like presentation247, but to difficulty with fine motor skills, such as handwriting, no improvement was reported in another patient with in children with PHP1A, leading to the need for occu- POH248. Finally, topical sodium thiosulfate has been pational therapy services in early childhood253. A high successfully administered in patients with hyperphos- incidence of carpal tunnel syndrome has been observed phataemic familial tumoural calcinosis or those with in patients with PHP1A and PPHP (67% of patients with hyperphosphataemia–hyperostosis syndrome with a PHP1A reported symptoms versus 15% of the general clinically and radiologically significant decrease of ectopic population)253. ossifications249. Preliminary data indicate that bisphospho- Additional skeletal features have been described in nates might also delay the progression of ectopic ossifica- different disorders of the PTH–PTHrP pathway, such as tions in patients with ossifications secondary to PHP or Madelung deformity72,128, spinal stenosis203,254–257, acro- related disorders250. Physical therapy and meticulous skin osteolysis, cortical irregularity of long bones and metadi- care remain the most important conservative approaches aphyseal enchondromata or short humerus and curved to preserve movement and to prevent cutaneous radius258, as well as other craniofacial peculiarities (typi- breakdown, respectively251,252. cal pear-shaped nose, long and flat philtrum, thin upper lip and receding chin) and phalangeal cone-shaped Recommendations epiphyses, resulting in clinodactyly259. Depending on 3.31. The presence of cutaneous bony plaques should the functional consequences, the patient might require be investigated by careful examination at each corrective orthopaedic surgery. visit in all patients with GNAS mutations, espe- Finally, many paediatric patients have a history of cially those with mutations on the paternal allele recurrent otitis media requiring a tympanostomy tube (POH and PPHP). Patients and families should be or tympanoplasty230.

Oral complications. Several oral manifestations have Patients with severe brachydactyly should receive been described in patients with PHP and related dis- formal evaluation of their fine motor skills as orders, including aplasia, thin enamel with enlarged well as be supplied with appropriate orthopaedic pulp chamber, hypoplasia, hypodontia, pulp calcifica- devices when indicated (that is, special shoes and tion, multiple carious teeth, multiple unerupted teeth or orthopaedic insoles) (A++). delayed tooth eruption, crowded anterior teeth, anterior 3.38. Specific multidisciplinary evaluation and therapy open bite, gingival hyperplasia, gingivitis with spontane- should be offered for rare orthopaedic or neuro- ous bleeding and pain88,260. Familial nonsyndromic pri- logical manifestations: spinal stenosis, bilateral mary failure of tooth eruption has also been described slipped capital femoral epiphyses, temporoman- in patients with mutations in PTHR1 and PTHrP88,261. dibular joint ankyloses, precocious scoliosis, Chiari malformation type 1 and cranial synostosis (A++). Recommendations 3.37. In addition to their use for diagnosis and character- Management of cognitive function ization of brachydactyly, further specific radiolog- Patients with PHP1A present with psychomotor and ical investigations should be reserved for patients cognitive abnormalities, which are defined as a history with specific clinical suspicion of orthopaedic mal- of developmental delay and learning disability, with formation or functional impairment, particularly reduced performance on the Wechsler intelligence in the presence of neurological signs or symptoms. scale24,42,262 and an increased incidence of psychiatric manifestations that might be the consequence of the disease or of long-term hypocalcaemia42,207. Box 1 | Future research directions In general, global retardation of developmental mile- diagnosis and natural history of the disorders stones, psychomotor retardation, delayed speech or the • extensive and systematic data on the following: need for an assistant teacher and extra school help are -- Growth pattern and final heights reported18,263,264. The performance IQ is more affected -- Bone maturation and timing of premature closure of the epiphysis than the verbal IQ24. Intellectual disability seems to be -- Pubertal development and gonadal function (including menopause), fertility and more prevalent in patients with PHP1A than in patients mineralization status with PPHP, which suggests that Gsα is imprinted in the -- Metabolic alterations and blood pressure brain24,214,229. The existence of neurological manifesta- -- Objective evaluation by standardized tests for cognitive and psychomotor tions or true neuropsychiatric phenotype due to organic impairment CNS alterations, and specifically Chiari malformation • identification of factors that contribute to the development and progression of ectopic type 1, should be considered38,207,265. Finally, the preva- bone formation lence and severity of cognitive impairment and develop- • Pursuit of initiatives for the development of a novel diagnostic classification mental delay vary among patients with acrodysostosis, Molecular diagnosis but a detailed clinical description is lacking5,6,14,63,266. • Frequency and associated phenotypes of the different molecular alterations • Development of testing methodology and validation of technologies for the Recommendation detection of partial methylation defects 3.39. Consider referral to a neuropsychologist for neuro- • Prevalence of multilocus imprinting disturbance (MLiD) and its effect on the phenotype cognitive and/or behavioural assessment at diagno- • identification of the prevalence and effects of genetic variants underlying sis or at preschool age, particularly in patients with methylation defects (affecting GNAS alone or MLiD) PHP1A and acrodysostosis due to PDE4D muta- • identification of the prevalence of molecular defects at the parathyroid hormone (PtH)– tions, and also if otherwise appropriate (for instance, parathyroid hormone-related protein (PtHrP) signalling cascade in a mosaic state if the patient presents with symptoms of cognitive • identification of new genes involved in the PtH–PtHrP signalling cascade and their impairment). Additional testing and support as association with as-yet-unresolved cases with PHP and related disorders required should also be considered (A++). • identification of the receptor pathway upstream of GNas that is associated with the development of ectopic ossification Malignancy risk Three case reports have described the coexistence of Follow-up and management PHP or a related disorder with malignancy: PHP1A and • Optimal calcium, phosphorus and PtH levels during active treatment for PtH resistance cerebellar pilocytic astrocytoma267, PHP1B and osteo- • Complications related to chronic hypocalcaemia, long-term PtH elevation and their sarcoma168 and POH with medulloblastoma82. However, treatment large-scale studies aimed at determining tumour risk in • indications, doses, efficacy and optimal timing for recombinant human growth PHP and related disorders have not been carried out. A hormone (rhGH) use and indication for puberty blockers Danish study has investigated all patients with a diagnosis • Contribution of reduced energy expenditure, decreased lipolysis, growth hormone- of PHP or a related disorder (clinical and/or genetically releasing hormone (GHrH) resistance and hyperphagia to the onset and degree of confirmed) to determine their mortality data and risk obesity of complications using data from the Danish National • Preclinical and clinical trials to investigate the benefit of post-surgical Patient Registry8. With a total of 60 cases, patients with bisphosphonates and of other candidate drugs (inhibitors of the Hedgehog signalling PHP or a related disorder were found to have an increased pathway and retinoic acid receptor- agonists) γ risk of neuropsychiatric disorders, infections, seizures and • Quality of life (QoL): both systematic evaluation with validated QoL questionnaires cataracts, whereas their risk of renal, cardiovascular (that is, the 36-item short Form survey (sF-36)) and validation of disease-specific and malignant disorders and fractures was similar to that questionnaires are needed of the general background population. The same results

were obtained when analysing only the subgroup with In addition, this group of disorders is caused by dif- genetically verified PHP or a related disorder. ferent and complex genetic and epigenetic defects, such An increased tumour risk might be hypothesized that establishing a correct molecular diagnosis might to exist in patients with PHP1B and MLID, mostly be difficult and time consuming for both patients and patients with MLID that are associated with Beckwith– their families and physicians. There is an urgent need to Wiedemann syndrome, a growth disorder characterized gather forces and cohorts of patients to implement regis- by embryonal tumours. However, the few data collected tries, to improve knowledge on the natural history of the to date did not report any tumours in patients with diseases, to better understand the bridges between these PHP1B and MLID92,104. clinically heterogeneous but still closely related diseases and, moreover, to develop new therapies. Recommendation This article, which is based on published evidence 3.40. There are no data to recommend a specific screen- and expert opinion, is the first international consensus ing for malignancies in PHP and related disorders statement for the diagnosis and management of PHP (A+++). and related disorders. The 67 recommendations apply to all patients with a clinical diagnosis, independently of Conclusions molecular confirmation. Nevertheless, in some patients, Patients with PHP and related disorders face a wide range the identification of the underlying genetic or epigenetic of problems from early childhood to adulthood. These defect might help clinicians to look for specific clinical include potentially severe alterations in mineral metab- manifestations with consequent appropriate manage- olism, which could be associated with seizures, other ment. A multidisciplinary approach is needed in most endocrine deficiencies due to hormone resistance that cases, and Table 4 summarizes the main interventions lead to hypothyroidism, hypogonadism and GH defi- as well as their timing. ciency, growth impairment independently of hormonal Given the lack of strong evidence-based data, status, ectopic ossifications with potential severe limita- particularly for management of these patients, tion of mobility, skeletal issues and cognitive and psych- international collaboration and long-term clinical omotor impairment. This highly heterogeneous clinical trials looking at the natural history, the diseases’ clas- picture renders a multidisciplinary approach mandatory sification and the outcome of treatments are urgently in these disorders, as very specialized expertise is required needed (Box 1). to manage each of the many clinical aspects and potential Published online xx xx xxxx complications of PHP and related disorders.

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